Proteins have become commercially important as drugs that are also generally called “biologicals”. One of the greatest challenges is the development of cost effective and efficient processes for purification of proteins on a commercial scale. Many methods are now available for large-scale preparation of proteins. However, crude products, contain also complex mixtures of impurities, which are sometimes difficult to separate from the desired product.
The health authorities request high standards of purity for proteins intended for human administration. In addition, many purification methods may contain steps requiring application of low or high pH, high salt concentrations or other extreme conditions that may jeopardize the biological activity of a given protein. Thus, for any protein it is a challenge to establish a purification process allowing for sufficient purity while retaining the biological activity of the protein.
Chromatography is an appropriate purification technology because it allows the separation of molecules having similar physico-chemical properties. Of the different type of chromatography, ion exchange chromatography, hydrophobic interaction chromatography, reverse phase chromatography and affinity chromatography are the most commonly used for industrial processes.
Ion exchange chromatographic systems have been used widely for separation of proteins primarily on the basis of differences in charge. In ion exchange chromatography, charged patches on the surface of the solute are attracted by opposite charges attached to a chromatography matrix, provided the ionic strength of the surrounding buffer is low. Elution is generally achieved by increasing the ionic strength (i.e. conductivity) of the buffer to compete with the solute for the charged sites of the ion exchange matrix. Changing the pH and thereby altering the charge of the solute is another way to achieve elution of the solute. The change in conductivity or pH may be gradual (gradient elution) or stepwise (step elution).
Anion exchangers can be classified as either weak or strong. The charge group on a weak anion exchanger is a weak base, which becomes de-protonated and, therefore, looses its charge at high pH. DEAE-cellulose is an example of a weak anion exchanger, where the amino group can be positively charged below pH ˜9 and gradually loses its charge at higher pH values. Diethylaminoethyl (DEAE) or diethyl-(2-hydroxy-propyl)aminoethyl (QAE) have chloride as counter ion, for instance.
A strong anion exchanger, on the other hand, contains a strong base, which remains positively charged throughout the pH range normally used for ion exchange chromatography (pH 1-14). Q-sepharose (Q stands for quaternary ammonium) is an example for a strong anion exchanger.
Cation exchangers can also be classified as either weak or strong. A strong cation exchanger contains a strong acid (such as a sulfopropyl group) that remains charged from pH 1-14; whereas a weak cation exchanger contains a weak acid (such as a carboxymethyl group), which gradually loses its charge as the pH decreases below 4 or 5. Carboxymethyl (CM) and sulfopropyl (SP) have sodium as counter ion, for example.
Chromatographic systems having a hydrophobic stationary phase have also been widely employed in the purification of proteins. Included in this category are hydrophobic interaction chromatography (HIC) and reversed phase liquid chromatography (RPLC). The physicochemical basis for separation by HIC and RPLC is the hydrophobic effect, proteins are separated on a hydrophobic stationary phase based on differences in hydrophobicity.
In HIC, generally, sample molecules in a high salt buffer are loaded on the HIC column. The salt in the buffer interacts with water molecules to reduce the solvation of the molecules in solution, thereby exposing hydrophobic regions in the sample molecules, which are consequently adsorbed by the HIC column. The more hydrophobic the molecule, the less salt needed to promote binding. Usually, a decreasing salt gradient is used to elute samples from the column. As the ionic strength decreases, the exposure of the hydrophilic regions of the molecules increases and molecules elute from the column in order of increasing hydrophobicity. Sample elution may also be achieved by the addition of mild organic modifiers or detergents to the elution buffer. HIC is reviewed e.g. in Protein Purification, 2d Ed., Springer-Verlag, New York, pgs 176-179 (1988).
In HIC, different chromatographic supports are available carrying various ligands. The ligands differ with respect to their hydrophobicity. Commonly used hydrophobic ligands are phenyl-, butyl- or octyl-residues.
Reverse phase chromatography is a protein purification method closely related to HIC, as both are based upon interactions between solvent-accessible non-polar groups on the surface of biomolecules and hydrophobic ligands of the matrix. However, ligands used in reverse phase chromatography are more highly substituted with hydrophobic ligands than HIC ligands. While the degree of substitution of HIC adsorbents may be in the range of 10-50 μmoles/mL of matrix of C2-C8 aryl ligands, several hundred μmoles/mL of matrix of C4-C8 alkyl ligands are usually used for reverse phase chromatography adsorbents.
Hydrophobic interaction chromatography and reverse phase chromatography are also distinct in that hydrophobic interaction chromatography is performed in aqueous solvent conditions and changes in ionic strength are used to elute the column. The protein typically binds in the native state via hydrophobic groups located on the surface of the protein, and the native state is retained during the elution conditions. In contrast to this, reverse phase chromatography utilizes a hydrophobic solvent (typically acetonitrile) and the binding of a ligand is a function of the phase partition between the hydrophobic nature of the solvent and column functional group. Proteins are typically denatured to some extent in such solvents and bind due to the hydrophobic nature of the entire polypeptide sequence. Since the majority of hydrophobic groups are located in the core of globular proteins, the binding is related to the extent of denaturation of the protein and the accessibility of these groups to the column functional groups.
Among the different techniques for protein purification, affinity chromatography deserves particular attention. It satisfies the requirement for ultra-high selectivity of the target protein from complex mixtures of impurities, thereby providing a better product quality.
Affinity chromatography relies on the biological functions of a protein to bind a ligand, i.e. a specific component, such as metal ions, peptides, chemical molecules, proteins, nucleic acids that is attached to a column matrix. This ligand can be immobilized or attached to a variety of matrixes, such as cellulose or agarose. The target protein is then passed through the column and bound to it via the ligand, while other proteins elute out. Purification of a target protein is usually achieved by passing a solution containing the target protein through the column that exhibits a high amount of attached or immobilized ligands. This is a very efficient purification method since it relies on the biological specificity of the target protein, such as the affinity of an enzyme for a substrate.
However, the variety of ligand available that can be attached to a matrix is enormous and the selection of the optimal ligand can not be easily inferred from one protein to the other. At least three limiting factors come into play, namely the different affinity of proteins to certain ligands, the immobilization of the ligand to the matrix in a sufficiently high amount and the potentially restricted accessibility of the ligand to the binding sites of the protein. Thus, it is extremely difficult to state a priori, which affinity chromatrography matrix will bind the target protein.
Interleukin-18 binding protein (IL-18BP) is a naturally occurring soluble protein that was initially affinity purified, on an IL-18 column, from urine (Novick et al. 1999). IL-18BP abolishes IL-18 induction of IFN-γ and IL-18 activation of NF-κB in vitro. In addition, IL-18BP inhibits induction of IFN-γ in mice injected with LPS.
The IL-18BP gene was localized to the human chromosome 11, and no exon coding for a transmembrane domain could be found in the 8.3 kb genomic sequence comprising the IL-18BP gene. Four isoforms of IL-18BP generated by alternative mRNA splicing have been identified in humans so far. They were designated IL-18BP a, b, c, and d, all sharing the same N-terminus and differing in the C-terminus (Novick et al 1999). These isoforms vary in their ability to bind IL-18 (Kim et al. 2000). Of the four human IL-18BP (hIL-18BP) isoforms, isoforms a and c are known to have a neutralizing capacity for IL-18. The most abundant IL-18BP isoform, isoform a, exhibits a high affinity for IL-18 with a rapid on-rate and a slow off-rate, and a dissociation constant (Kd) of approximately 0.4 nM (Kim et al. 2000). IL-18BP is constitutively expressed in the spleen, and belongs to the immunoglobulin superfamily. The residues involved in the interaction of IL-18 with IL-18BP have been described through the use of computer modelling (Kim et al. 2000) and based on the interaction between the similar protein IL-1 with the IL-1R type I (Vigers et al. 1997).
IL-18BP is constitutively present in many cells (Puren et al. 1999) and circulates in healthy humans (Urushihara et al. 2000), representing a unique phenomenon in cytokine biology. Due to the high affinity of IL-18BP to IL-18 (Kd=0.4 nM) as well as the high concentration of IL-18BP found in the circulation (20 fold molar excess over IL-18), it has been speculated that most, if not all of the IL-18 molecules in the circulation are bound to IL-18BP. Thus, the circulating IL-18BP that competes with cell surface receptors for IL-18 may act as a natural anti-inflammatory and an immunosuppressive molecule.
IL-18BP has been suggested as a therapeutic protein in a number of diseases and disorders, such as psoriasis, Crohn's Disease, rheumatoid arthritis, psoriatic arthritis, liver injury, sepsis, atherosclerosis, ischemic heart diseases, allergies, etc., see e.g. WO 99/09063, WO 01/07480, WO 01/62285, WO 01/85201, WO 02/060479, WO 02/096456, WO 03/080104, WO 02/092008, WO 02/101049, WO 03/013577. Given that IL-18BP is suggested as a therapeutic protein for administration e.g. to humans, there is an unmet need for adequate amounts of IL-18BP in sufficiently high purity.
A purification process for IL-18BP has been described in WO 2005/049649. However, this process does not comprise a step of affinity chromatography.
Thus, an alternative purification process resulting in IL-18BP in good purity and in high yield is desirable.